The invention concerns mass spectrometers, and more specifically tandem time-of-flight mass spectrometers.
Mass spectrometry comprises a broad range of instruments and methodologies that are used to elucidate the structural and chemical properties of molecules, to identify the compounds present in physical and biological matter, and to quantify the chemical substances found in samples of such matter. Mass spectrometers can detect minute quantities of pure substances (regularly, as little as 10xe2x88x9212 g) and, as a consequence, can identify compounds at very low concentrations (one part in 10xe2x88x9212 g) in chemically complex mixtures. Mass spectrometry is a powerful analytical science that is a necessary adjunct to research in every division of natural and biological science and provides valuable information to a wide range of technologically based professions, such as medicine, law enforcement, process control engineering, chemical manufacturing, pharmacy, biotechnology, food processing and testing, and environmental engineering. In these applications, mass spectrometry is used to identify structures of biomolecules, such as carbohydrates, nucleic acids and steroids; sequence biopolymers, such as proteins and oligosaccharides; determine how drugs are used by the body; perform forensic analyses, such as confirmation and quantiation of drugs of abuse; analyze environmental pollutants; determine the age and origins of geochemical and archaeological specimens; identify and quantitate components of complex organic mixtures; and perform ultrasensitive, multi-element analyses of inorganic materials, such as metal alloys and semiconductors.
Mass spectrometers measure the masses of individual molecules that have been converted to gas-phase ions, i.e., to electrically charged molecules in a gaseous state. The principal parts of a typical mass spectrometer are the ion source, mass analyzer, detector, and data handling system. Solid, liquid, or vapor samples are introduced into the ion source where ionization and volatilization occur. The form of the sample and the size and structure of the molecules determine which physical and chemical processes must be used in the ion source to convert the sample into gas-phase ions. To effect ionization, it is necessary to transfer some form of energy to the sample molecules. In most instances, this causes some of the nascent molecular ions to disintegrate (either somewhere in the ion source or just after they exit the ion source) into a variety of fragment ions. Both surviving molecular ions and fragment ions formed in the ion source are passed on to the mass analyzer, which uses electromagnetic forces to sort them according to their mass-to-charge ratios (m/z), or a related mechanical property, such as velocity, momentum, or energy. After they are separated by the analyzer, the ions are successively directed to the detector. The detector generates electrical signals, the magnitudes of which are proportional to the number of ions striking the detector per unit time. The data system records these electrical signals and displays them on a monitor or prints them out in the form of a mass spectrum, i.e., a graph of signal intensity versus m/z. In principle, the pattern of molecular-ion and fragment-ion signals that appear in the mass spectrum of a pure compound constitutes a unique chemical fingerprint from which the compound""s molecular mass and, sometimes, its structure can be deduced.
Tandem Mass Spectrometers
The utility of a mass spectrometric analysis can be significantly enhanced by performing two (or more) stages of mass analysis in tandem. A two-stage instrument is referred to herein as an MS/MS instrument. An MS/MS instrument performs two (or more) independent mass analyses in sequence. In the most frequently used mode of MS/MS, ions of a particular m/z value are selected in the first stage of mass analysis (MS1) from among all the ions of various masses formed in the source. The selected ions (referred to as precursor ions) are energized, usually by collision with a neutral gas molecule, to induce dissociation. The product ions of these dissociations are sorted into a product-ion mass spectrum in the second stage of mass analysis (MS2). If the sample is a pure compound and fragment-forming ionization has been used, individual fragment ions originating in the ion source can be selected as precursor ions; their product-ion spectra (which may be thought of as mass spectra within a mass spectrum) provide much additional structural information about the compound. If the sample is a mixture and nonfragment-forming ionization is used to produce predominantly molecular ions, the second stage of mass analysis can provide an identifying mass spectrum for each component in the mixture.
Independent operation of each stage of mass analysis makes possible other MS/MS operations based on changes in mass, charge, or reactivity and on the ability of the mass spectrometer to define those changes. MS/MS also can be used to substantially improve signal-to-background ratios and, therefore, sensitivity by eliminating interferences in certain types of operations when the ion signal at the m/z of interest is produced by more than one compound. Increasingly, MS/MS is being used to probe more precisely into problems of ion structure as well as to increase resolution in analyses of complex mixtures.
Time-of-Flight Mass Spectrometers
At present, the most widely used mass analyzers are magnetic sectors, quadrupole mass filters, quadrupole ion traps, Fourier-transform ion-cyclotron resonance cells, and time-of-flight (TOF) tubes. TOF mass analyzers are fundamentally the simplest and the least expensive to manufacture. They separate ions by virtue of their different flight times over a known distance. To create these different times, an ensemble of ions of like charge are accelerated to essentially equal kinetic energies and, in a brief burst, released from the ion source into the flight tube. Since an ion""s kinetic energy is equal to xc2xd mv2 (where m is its mass and v its velocity) and all ions of like charge have substantially the same energy, light ions will have greater velocities and, consequently, shorter flight times to the detector than heavy ions. The m/z values of each set of ions contained in a given burst out of the ion source can be determined by measuring their successive transit times from the ion source through the flight tube to the detector (typically several tens of microseconds).
A TOF mass spectrometer is unique in that its m/z-range is theoretically unlimited, and its mass spectra are not produced by scanning. Moreover, it is a relatively simple, inexpensive instrument to manufacture and operate. These three features account in large part for the major role TOF instruments have played in the rapidly expanding usage of mass spectrometry in molecular-biological research and biotechnology.
With the other four commonly used mass analyzers, the settings of one or more parameters determines the m/z of the ions that are allowed to pass to the detector. In order for ions with a different m/z to be detected, these settings must be increased or decreased. Ultimately, some fundamental or practical characteristic of the mass analyzer limits the extent to which its m/z-determining parameters can be changed to accommodate analysis of increasingly larger ions. In a TOF mass analyzer, increasingly larger ions simply take increasingly longer times to reach the detector, and there is no limit to the length of time that can be measured. Thus, TOF mass analyzers are especially useful for the analysis of large biological molecules.
Scanning denotes a continuous increasing or decreasing of a mass analyzer""s m/z-determining parameters over a predetermined range so that ions over a corresponding range of m/z-values can be detected in succession. The analytical efficiency of a mass spectrometric analysis is greatly reduced by scanning because, while the ions of one particular m/z are being detected, the ions of all other m/z-values released from the ion source are being irretrievably lost in the instrument. With TOF mass analyzers, by contrast, all of the ions released in an ion-burst from the source are detected and recorded without changing any instrumental parameters. Consequently, TOF mass spectrometers are particularly sensitive instruments.
TOF mass spectrometers may be constructed using relatively simple components, such as accelerators, ion reflectors, and ion detectors. Moreover, TOF mass spectrometers are relatively simple to operate because they are stable in operation and the components require minimal tuning. Because of their relatively simple construction and operation, TOF mass spectrometers are relatively inexpensive to construct and operate.
Referring to FIG. 1, a typical time-of-flight mass spectrometer includes an ion source, a drift region, and a detector. Ion sources have two components: an ionization chamber and an ion extractor/accelerator. A sample is received in the ionization chamber, volatilized if necessary, and ionized (usually by some is energetic process). An ionization process that is particularly suited for large biological molecules is matrix-assisted laser desorption/ionization (MALDI). MALDI requires that the sample be dispersed within a matrix of solid, crystalline material. A laser is focused on the sample to volatilize and ionize the sample. The process also may cause a portion of the resulting ions to dissociate into smaller fragment ions. Those of ordinary skill in the art are familiar with the construction and operation of MALDI sources.
Focusing Ions
In general, ions formed in a TOF ion source have different times of formation, initial positions and velocities. Without some form of correction, these variations in the ions"" initial mechanical properties diminish the resolution of the mass-dependant bands that are later detected. The uncertainties associated with these variations can be corrected one at a time, but it is very difficult with a single device to correct for two or more of them simultaneously. Wiley and McLaren developed a two-stage ion source that, with certain restrictions, can force ions having the same mass-to-charge but different initial positions or velocities to arrive nearly simultaneously at a particular plane some distance downstream of the ion source. [Wiley, W. C.; McLaren, I. H.; Review of Scientific Instruments, Vol. 26:12, pp. 1150-1157 (1955), which is incorporated herein by reference]. The position of this plane, which is referred to as the space focal plane, is uniquely defined by the ion source""s geometry, and the voltage applied to the ion source""s electrodes. [Potter, R. J.; xe2x80x9cTime-of-Flight Mass Spectrometry,xe2x80x9d American Chemical Society, Washington, D.C., (1997), which is incorporated herein by reference.] In a Wiley/McLaren two-stage source, the ionization chamber (or ionization region) is separated from the accelerating region by a plane grid. The electric field in the ionization region (or first stage) is made smaller than in the accelerating region electric field by adjusting the voltages applied to the sources backing plate and to the grid separating the two regions. Ions move from the ionization region, into the acceleration region, and out of the source under the influence of these electric fields. Ions can be generated in the ionization chamber or injected into it. After a short time delay the electric field in the ionization region is switched on. This electric field moves the ions out of the first stage into the second stage where they are accelerated to greater velocities.
By means of time-lag focusing, the two-stage ion source is able to correct for variation in the initial velocities of the ions providing they are formed in, or nearly in, a plane that is parallel to the backing plate and the dividing grid. During the lag period xcfx84, the ions spread out in the ionization chamber in accordance with their velocities at the time of their formation. Those ions that are closer to the backing plate when the electric field is switched on (lagging ions) are accelerated over a longer distance before entering the second stage than ions farther from the backing plate (leading ions). Thus, the lagging ions receive more kinetic energy from the first electric field than the leading ions. Consequently, the lagging ions eventually catch up with leading ions of the same m/z. At the plane where the lagging ions catch the leading ions of the same m/z, the ions are said to be energy-focused. The time lag energy focal plane of an ion source coincides with that ion source""s space focal plane. The distance from the exit of the accelerating region to the plane where space focusing occurs is referred to as the space-focal length f. Under a fixed set of conditions, f is mass dependent so ions of different masses will have different focal lengths. In a given mass spectrometer, the distance from the exit of the accelerating region to any ion-optical component downstream of the ion source, e.g., velocity selector, post velocity selection accelerator, ion reflector, or ion detector, is fixed by the instrument""s geometry. The focal length for ions of any particular mass can always be made equal to any of these fixed distances by adjusting the ratio Ea/Ee of the electric fields in the accelerating region (Ea) and the extraction region (Ee) and, in the case of time-lag-focusing, the delay or lag time xcfx84. Because the dimensions of these regions are fixed for a given ion source, Ea/Ee is varied by changing the voltages applied to the repeller plate and the extractor/accelerator grid that separates the two regions.
Tandem Time-of-Flight Mass Spectrometers
TOF mass spectrometry""s steadily growing range of application in biomolecular analysis has prompted several attempts to develop tandem TOF instruments. Two approaches have been taken: 1) coupling two independently usable mass spectrometers together; and 2) using velocity selection as the basis for the first stage of mass spectrometry in the tandem sequence of operations. The first is classical and general to all forms of mass spectrometry. The second is recent and specific to time-of-flight mass spectrometers. The classical approach to constructing a tandem TOF mass spectrometer has so far produced three instrument forms: 1) a high-resolution TOF mass analyzer coupled to a high resolution TOF mass analyzer (TOF/TOF); 2) a double-focusing sector mass analyzer coupled to a high resolution TOF mass analyzer (Sector/TOF); and 3) a quadrupole mass filter coupled to a high- resolution TOF mass analyzer (Q/TOF). These instruments produce mass spectra that exhibit unit mass-resolution Q/TOF) or better (TOF/TOF and Sector/TOF) in the first stage of mass analysis (MS1). In the cases of TOF/TOF and Sector/TOF, the gain in MS1-resolution is offset by low sensitivity because, in instances where PSD is the predominant fragmentation process, very few precursors reach the collision cell and the second stage of mass analysis (MS2) or, in instances where the collision cell must be used to induce fragmentation, the collisional dissociation process interferes with the timing and transmissions of the MS2-TOF. Either way, the performance and, hence, utility of these tandem instruments are degraded.
Q/TOF instruments are manufactured by two companies: Micromass Ltd., Floats Road, Wythenshawe, Manchester M23 9LZ, UK; and PE SEIEX, Concord, Ontario, L4K 4V8, Canada. Unfortunately, the gain in the Q/TOF""s MS1-performance is offset by the fact that only low energy ions (10-40 eV) can be analyzed by MS1. This restriction excludes ions produced by MALDI, which is one of the most versatile and widely used ionization methods.
The velocity selection approach to configuring a tandem TOF mass spectrometer takes advantage of the fact that product-ions resulting from metastable or induced decompositions in a time-of-flight tube retain to the first order the velocity of their precursors (parent ions). This approach is appealing because it offers a means to preserve much of the single TOF mass analyzer""s sensitivity and simplicity. The reality to date, however, is that tandem TOF configurations based strictly on velocity selection in MS1 do not. achieve analytically useful resolution in MS1, MS2, or both. To the extent they have succeeded, such instruments have sacrificed one or more of the TOF mass analyzer""s three advantageous features: theoretically unlimited mass range, recording without scanning, and simple and inexpensive construction and operation.
The post-source decay (PSD) method introduced by Kaufmann et al. [Kaufmann, R.; Spengler, B.; Lxc3xctzenkirchen, F. Rapid Communications in Mass Spectrometry, 7:902-10 (1993); Kaufmann, R., Kirsch, D., Spengler, B., International Journal of Mass, Spectrometry and Ion Processes, Vol. 131:355-85 (1994)], presently is the most widely used form of tandem TOF mass spectrometry based on velocity selection. In addition to being hampered by the general technological failings of current velocity selection configurations, PSD suffers the additional drawback of being inextricably coupled to the MALDI ionization technique. More generally, PSD relies exclusively on the statistically governed processes of metastable decomposition and random gas-phase collisions during flight to produce fragment ions for analysis in MS2. Each of these phenomena tend to be promoted in several compounds under MALDI conditions; hence, the almost inseparable association between PSD and MALDI. When metastable and gas-phase decompositions are not promoted by MALDI (as is often the case with important compounds), PSD provides no recourse to any other means for producing fragment ions.
Tandem time-of-flight mass spectrometers that use velocity selection as the basis for MS1 employ ion deflectors as gates for selecting a band of ions having a desired m/z. The classical geometry for such a gate uses a pair of parallel plates to define a uniform electric field perpendicular to the flight path of the ions. When the electric field is on, it deflects ions that enter the gate so that they do not reach the detector. When the ions to be selected reach the gate, the electric field is switched off to allow those ions to pass through. As soon as the desired ions have passed through the gate, the electric field is switched back on so that ions subsequently entering the gate also are deflected. Parallel plate velocity selectors have relatively large capacitances (5-10 pf); therefore, they are difficult to switch on and off in less than 30-50 ms; shorter switching times are necessary to produce high enough resolving powers (m/xcex94m, where m is the mass of the select ions and xcex94m is the range of masses selected) to be effective in tandem TOF mass spectrometers.
A more effective gate geometry than parallel plates is an arrangement of parallel wires. Wires have less capacitance than plates (xe2x89xa6pf) and can be switched in 5-10 nanoseconds; however, they impart less deflection impulse to passing ions.
Some have achieved slightly better resolutions using dual deflector velocity selectors. With such velocity selectors, the first deflector is initially on so as to deflect ions passing through it, and the second deflector located downstream of the first is initially off. When the desired ions approach, the first deflector is switched off to allow them to pass through without being deflected. As soon as the selected ions pass through the second deflector, again without being deflected, the second deflector is switched on. Although it is possible to make the time between when the first deflector is switched off and the second deflector is switched on smaller than the time it takes to turn a single deflector off and then back on again, dual deflectors operated in this manner have not been able to provide high enough resolving powers to operate very effectively as velocity selectors in tandem TOF mass spectrometers.
If one can ignore the tiny amount of energy released when a molecular ion decays in the flight tube of a TOF instrument, conservation of energy and momentum require that the fragments of the decay (both charged and neutral) continue flight with exactly the same velocity their parent had. Therefore, when particles are selected in the flight tube on the basis of velocity, the selected group can contain nondissociated precursors, fragments of dissociated precursors, or both. If the group does contain nondissociated precursors and some or all of these do dissociate in the length of flight tube remaining between MS1 and MS2, the fragments of those decays would simply continue to fly along with the selected group. Hence, the velocity-selected group of particles enters MS2 of a tandem TOF instrument as a spatially and temporally confined band. At this stage, the kinetic energies of the fragments are proportional to their masses, and the maximum kinetic energy for any fragment equals that of the nondissociated precursor ions, qVion source (where q is the charge carried by a precursor ion). For example, the kinetic energies of the fragments of a 20 keV, 3000 Da precursor ion would essentially cover the range 0-20 keV (1 Da={fraction (1/12)} of the mass of a single atom of 12C).
In order to separate particles with identical velocities but different energies, the particles must be accelerated. In current tandem TOF instruments, ion reflection is used to accomplish this task. The selected group (fragments and ions that have not fragmented) are directed into an ion reflector (ion mirror or reflectron), which is the main component of MS2. A typical linear-field reflectron creates a highly uniform axial electric field that accelerates the ions in a direction exactly opposed to the direction of their entry. Thus, ions entering the reflectron at a particular angle of incidence xcfx86 are gradually slowed to a stop and then gradually speeded up in the direction from which they came so that they exit the mirror at a reflected angle exactly equal to xcfx86. Providing xcfx86 is not too large ( less than 2 or 3xc2x0), a typical linear-field reflectron is able to focus ions at a space focal plane, located some distance from the exit of the reflectron where a detector is normally mounted. The reflectron will only focus ions of the same mass at its space focal plane if those ions were spaced-focused at its object plane prior to entering it. The neutral fragments in the selected group are not acted upon by electric fields and, therefore, pass straight though the reflectron; the neutrals can be recorded by a detector placed beyond the rear of the reflectron.
Ions entering the reflectron with more kinetic energy will penetrate deeper into the reflectron than ions entering the reflectron with less kinetic energy. Because fragment ions always have less energy than their parent ions, fragment ions exit the reflectron more quickly and reach the detector sooner than their precursors. Not all of these fragment ions will be space-focused at the plane of the detector. Only those ions that require 85-95% of the length of the ion mirror to be reflected will be space-focused when they arrive at the detector. Ions with less energy (lighter ions) will penetrate less deeply into the reflectron and will not be space-focused at the detector. Thus, dispersion by ion reflection can only produce a complete mass spectrum of the fragment ions, similar to one that would be produced by a tandem mass spectrometer, when the voltage of the reflectron is stepped. A partial spectrum is produced with each step, and the partial spectra are assembled in order to produce the complete spectrum. For example, a spectrum covering the mass-range of the product ions of the 20 keV, 3000 Da precursor ion described above can be acquired on existing instruments equipped with a single stage, linear-field reflectron by stepping the reflectron""s voltage setting 7-10 times (10-14 times with a double stage ion-reflectron) and recording a segment of the spectrum at each setting. The TOF mass analyzer""s highly valued nonscanning feature is sacrificed when it becomes necessary to resort to this time-consuming, sample-wasting, manual stepping procedure.
Cotter""s U.S. Pat. No. 5,464,985 discloses a tandem TOF mass spectrometer. Cotter""s spectrometer selects precursor ions in MS1 according to their velocity and disperses the fragment ions and nondissociated precursor ions with a reflectron. Cotter avoids the linear reflectrons limited mass range problem by using a curved-field reflectron to record an entire spectrum at a single voltage setting. The curved-field reflectron uses a nonlinear axial electric field to achieve focusing across a wide mass range of product ions without stepping or otherwise changing the reflectron""s voltage setting. Despite its elegant conception, the curved-field reflectron is not practical. The curved-field reflectron, which consists of 86 ring elements (instead of 30 or so for a simple linear-field reflectron) connected by 85, 20-turn, 2 Mxcexa9 potentiometers (instead of 29 or so uniform fixed resistors), is difficult to construct, is difficult to tune (each potentiometer must be painstakingly adjusted to precisely replicate the required curvature in the axial component of its electric field), is difficult to maintain in tune because of non-uniform drift in the potentiometers"" settings, and has low ion-transmission because of the defocusing action of the unavoidable radial component of its electric field. Thus, the TOF mass analyzer""s stable operation and simple, low-cost construction are sacrificed in Cotter""s spectrometer.
The present invention overcomes the problems discussed above. The invention provides a tandem time-of-flight mass spectrometer that includes an ion source, an ion selector downstream of the ion source, a dissociation cell downstream of the ion selector, an ion accelerator downstream of the dissociation cell, an ion reflector (ion mirror or reflectron) downstream of the accelerator, a detector that records neutrals and ions transmitted through the reflectron, and a detector that records ions reflected by the reflectron. The spectrometer of the present invention is stable in operation, simple in construction, has high resolving powers, and is able to focus product ions at a space focal plane located at a detector.
In one embodiment of the invention, the mass spectrometer includes an ion source, a velocity selector downstream of the ion source, a dissociation cell downstream of the velocity selector, an ion accelerator downstream of the dissociation cell, the accelerator being capable of focusing ions at a first space focal plane, a reflectron downstream of the accelerator, the reflectron defining an object plane located at the first space focal plane, and the reflectron being capable of focusing ions at a second space focal plane, and an ion detector located at the second space focal plane. The position of the accelerator in this embodiment is particularly advantageous because it accelerates the product ions in a manner that minimizes the effects of the uncertainties of ions leaving the dissociation cell, allows the ions to subsequently separate according to their m/z ratios, and allows the ions to be easily focused at a detector.
The invention also provides a method for producing a mass spectrum that includes accelerating a set of ions to give the set of ions mass-dependant velocities, selecting a subset of the set of ions based on their velocities, inducing dissociation of a fraction of the selected subset of ions if necessary, and detecting the subset of ions. In one embodiment, the method of producing a mass spectrum includes ionizing a material to produce a set of ions. The set of ions is then accelerated to a constant energy so that each ion has a mass-dependant velocity. The accelerated set of ions is allowed to drift along a flight path so that subsets of ions within the set of ions that have different velocities become spatially separated along the flight path. The set of ions, except a select subset of the ions within a narrow, select velocity range, are subsequently deflected from the flight path. In one embodiment, a fraction of the subset of select ions are then induced to dissociate, and the resulting fragment ions along with the remainder of nondissociated ions in the original subset of ions are accelerated along the flight path to mass-dependant velocities, allowed to drift along the flight path so that ions of different velocities spatially separate, and detected at a location along the flight path. The acceleration of the subset of ions along the flight path after the ions dissociate allows the ions to subsequently separate according to their m/z ratios and allows them to be easily focused onto a detector. In one embodiment, the subset of ions are accelerated by an electric field that is switched on once all the ions have moved into the space between the electrodes that create the field, so that the subset of ions will focus at a space focal plane.
The invention also provides a novel velocity selector that is able to attain high resolving powers. A high resolving power corresponds to the selection of a very small range of velocities and, therefore a very small range of masses. An embodiment of the velocity selector includes a first ion deflector having multiple, electrically conductive strips that define multiple channels. The strips include alternate positive voltage strips connected to a first positive voltage source, and alternate negative voltage strips connected to a first negative voltage source. A second ion deflector is in series with the first ion deflector. The second ion deflector includes multiple, electrically conductive strips defining multiple channels. The strips include alternate positive voltage strips connected to a second positive voltage source, and alternate negative voltage strips connected to a second negative voltage source. The strips can impart sufficient deflection impulse to passing ions, and yet their low capacitance enables short switching times.
The invention also provides a method for selecting a subset of ions from a set of ions. In one embodiment, the method includes accelerating a set of ions in the ion source so the ions have varying velocities, and allowing the set of ions to move along a flight path so that ions of different velocities spatially separate along the flight path. A voltage is applied across a first ion deflector positioned along the flight path so as to deflect ions passing through the first deflector in a first direction away from the flight path. The voltage across the first deflector is switched off so that a subset of ions having a select range of velocities is deflected less in the first direction than preceding ions, and a voltage across a second ion deflector arranged downstream of the first deflector switched on so as to deflect ions passing through the second deflector in a second direction, which is exactly opposite the first direction. The voltages and switching times are such that the second deflector deflects the subset of ions with the select velocity back along the flight path but deflects ions following the subset of ions with the select velocity away from the flight path in the second direction. The method of ion selection produces particularly high resolving powers because it allows the electric fields generated by the deflectors to interact dynamically with the subset of ions with the select velocity as they pass through the deflectors.